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NOTE: As a convenient point of reference for readers, the following methodology describes the specific culture conditions and materials required for the measurement of Fe bioavailability from 20 experimental samples, plus the required quality controls, in a run of the bioassay. Increasing the number of samples beyond this capacity is not recommended due to the time required for various cell culture and in vitro digestion steps within the bioassay.
1. Choosing the amount of samples
<ol>
	<li>For solid or liquid foods, determine an amount of food that can be considered representative of the sample to be tested.
	<ol>
		<li>In testing for Fe bioavailability from a bean variety, use 100-150 g of bean seed and process this amount to a homogeneous sample.</li>
		<li>For liquid samples such as fortified juices, milk products, and sports beverages, ensure that the food is well mixed prior to sampling. 
		NOTE: The amount of bean seed material mentioned above is essential to account for the inherent differences between seeds of this staple crop.</li>
	</ol>
	</li>
</ol>
2. Preparation of samples
<ol>
	<li>Rinse off soil and dust from any food sample with distilled-deionized water before processing.</li>
	<li>Process the appropriate amount of sample as per the experimental objectives, such as by cooking method and milling. 
	NOTE: For cooking and processing, it is critical to use cookware and equipment that is not a potential source of contaminant Fe. Stainless steel equipment does not contaminate; however, equipment such as stone mill grinders, cast iron cookware, and any non-stainless steel equipment containing Fe can add significant amounts of contaminant Fe. A standard stainless steel coffee grinder is often adequate for grinding.</li>
	<li>Lyophilize and grind to a homogeneous powder. 
	NOTE: Once homogenized, research has shown that three independent replications of analysis are adequate for each food being measured.
	<ol>
		<li>If sample homogeneity is difficult to achieve, revise the formulation or processing of the product. If this is not possible, add replications if the non-homogeneity is not severe.</li>
		<li>For most homogeneous solid foods, use 0.5 g of lyophilized sample per replicate. If necessary, use up to 1.0 g of sample per replicate, but check if more than 0.5 g yields a benefit in the degree of response. 
		NOTE: Amounts higher than 0.5 g of solid foods may clog the dialysis membrane (see below).</li>
		<li>Use 1-2 mL of liquid samples. 
		NOTE: Lyophilization is often not necessary for liquid samples.</li>
	</ol>
	</li>
</ol>
3. Caco-2 cell culture
<ol>
	<li>Stock cultures
	<ol>
		<li>Acquire Caco-2 cells from a certified supplier.</li>
		<li>Culture the cells from stock vials at 37 &#176;C in an incubator with a 5% CO2 air atmosphere (constant humidity) using Dulbecco&#39;s Modified Eagle&#39;s Medium (DMEM) supplemented with 25 mM HEPES (pH 7.2), 10% (v/v) fetal bovine serum (FBS), and 1% antibiotic-antimycotic solution.</li>
		<li>Once sufficient cells are available, usually after 7-10 days of culture, seed the cells in non-collagen-coated flasks at a density of 30,000 cells/cm2.</li>
		<li>Choose the flask size depending on the number of cells available and needed for seeding multiwell plates. 
		NOTE: In general, the T225 (225 cm2) flasks work best for experiments where 11 multiwell (6-well; 9.66 cm2/well) plates are used (10 plates for sample comparisons, 1 plate for quality controls) per bioassay.</li>
		<li>Grow cells in flasks for 7 days, changing the medium every other day, and use on the 7th day for seeding the multiwell plates. 
		NOTE: It is recommended to use a passage range of no more than 10-15 passages from when the cells are started from stock culture and subsequently used in a series of experiments. Cell culture passages should be limited as a broad range of passages can result in adaptive changes in the cell line and, thus, variability in the response of the model.</li>
	</ol>
	</li>
	<li>Cell culture on multiwell plates
	<ol>
		<li>Seed the Caco-2 cells at a density of 50,000 cells/cm2 in 6-well collagen-coated plates. 
		NOTE: This step usually works best if done on a Wednesday. The following steps will make it evident why this day of the week is optimal.</li>
		<li>Grow the cells for 12 days at 37 &#176;C in an incubator with a 5% CO2 air atmosphere (constant humidity) using DMEM supplemented with 25 mM HEPES (pH 7.2), 10% (v/v) FBS, and 1% antibiotic-antimycotic solution. 
		NOTE: Culturing the cell monolayers longer than 12 days can result in cell overgrowth. Previous research has clearly shown that, under these conditions, at 12 days post-seeding, the cell monolayer is mature, attached well to the plate, and optimal in the consistency of response. Growing the cells longer, such as up to 19-21 days, results in cell overgrowth, and the media rapidly depletes in nutrients, resulting in unhealthy monolayers.</li>
		<li>During the 12-day period, change the medium at least every 2 days on a consistent daily schedule.</li>
		<li>On the 12th day post seeding, replace the culture medium with 2 mL of Minimum Essential Medium (MEM [pH 7]) supplemented with 10 mM PIPES (piperazine-N,N&#39;-bis-[2-ethanesulfonic acid]), 1% antibiotic-antimycotic solution, hydrocortisone (4 mg/L), insulin (5 mg/L), selenium (5 &#956;g/L), triiodothyronine (34 &#956;g/L), and epidermal growth factor (20 &#956;g/L). 
		NOTE: If seeding was started on a Wednesday, then the 12th day would be a Monday.</li>
		<li>On the following day (i.e., day 13), remove the MEM and replace it with 1 mL of MEM (pH 7). 
		NOTE: This step would occur on a Tuesday. This is the day when the bioassay begins; thus, the 13-day schedule yields the advantage of a consistent weekly schedule, allowing the bioassay to be conducted consistently on the same weekday.</li>
	</ol>
	</li>
</ol>
4. In vitro digestion
<ol>
	<li>Preparation of insert rings
	<ol>
		<li>Create a sterilized insert ring using a silicone O-ring fitted with an acid-washed dialysis membrane (Figure 1A). 
		NOTE: Prepare inserts 1 day in advance (i.e., Monday, day 12) of the day of the bioassay and store in 18 M&#937; water at 4 &#176;C until ready to use.</li>
		<li>On the day of the bioassay (Tuesday, day 13), remove the inserts from the refrigerator, drain, and replace the water with 0.5 M HCl. Leave it at room temperature in a laminar flow hood for at least 1 h prior to use. 
		NOTE: This step should be done prior to removing the MEM from the culture plates. Acid washing of the membrane serves to remove possible contaminating Fe and sterilizes the insert ring and membrane.</li>
		<li>Drain the 0.5 M HCl from the inserts and rinse with sterile 18 M&#937; water. Store in sterile 18 M&#937; water at room temperature in a laminar flow hood until ready to use.</li>
		<li>Insert a ring into each well of the 6-well plates with Caco-2 cells, thereby creating a two-chamber system. Return the plates with the inserts to the incubator. 
		NOTE: This step should be done just after fresh 1.0 mL of MEM is added to each well (see Step 3.2.5.).</li>
	</ol>
	</li>
	<li>Preparation of pepsin solution
	<ol>
		<li>On the day of the experiment, prepare the pepsin solution by dissolving 0.145 g of pepsin in 50 mL of 0.1 M HCl. Shake the solution gently on a platform shaker for 30 min at room temperature.</li>
	</ol>
	</li>
	<li>Preparation of pancreatin-bile solution
	<ol>
		<li>On the same day as the experiment, prepare 0.1 M NaHCO3 by dissolving 2.1 g of NaHCO3 in 250 mL of 18 M&#937; water.</li>
		<li>Mix 0.35 g of pancreatin and 2.1 g of bile extract in 175 mL of 0.1 M NaHCO3.</li>
		<li>Once the pancreatin and bile extract are solubilized, add 87.5 g of a weak cation exchange resin (see the Table of Materials) and mix by shaking for 30 min at room temperature.</li>
		<li>Pour the slurry into a large column and collect the eluate.</li>
		<li>Elute the column with an additional 70 mL of 0.1 M NaHCO3, collecting this volume into the pancreatin bile solution. 
		NOTE: The purpose of the resin is to remove contaminant Fe commonly found in the pancreatin-bile extracts.</li>
	</ol>
	</li>
	<li>Initiate in vitro digestion.
	<ol>
		<li>Weigh out the sample in a sterile 50 mL centrifuge tube (polypropylene), followed by the addition of 10 mL of physiological saline at pH 2, containing 140 mM NaCl and 5 mM KCl.</li>
		<li>Initiate the gastric digestion process by adding 0.5 mL of the prepared porcine pepsin solution to the sample and incubate on a rocking shaker at a low, gentle setting for 1 h at 37 &#176;C.</li>
		<li>Following this period, initiate the intestinal digestion process of each sample by adjusting the pH to 5.5-6.0 with 1.0 M NaHCO3.</li>
		<li>Add 2.5 mL of the pancreatin-bile solution to each sample tube and adjust the pH to 6.9-7.0 with 1.0 M NaHCO3.</li>
		<li>Once the pH is adjusted, equalize the volume in each tube using 140 mM NaCl, 5 mM KCl (pH 6.7) solution, measuring the weight of the tube with a target value of 15 g. 
		NOTE: For some foods, one may need to bring the total volume to 16 g or 17 g, depending on the buffering capacity of the foods.</li>
		<li>Transfer 1.5 mL of each intestinal digest into the upper chamber (i.e., containing the insert ring with the dialysis membrane) of a corresponding well of the 6-well culture plate containing the Caco-2 cells (Figure 1B).</li>
		<li>Replace the plate cover and incubate at 37 &#176;C (5% CO2 air atmosphere) on a rocking shaker at 6 oscillations/min for 2 h.</li>
		<li>Remove the insert ring with the digest and add an additional 1 mL of MEM (pH 7) to each well.</li>
		<li>Return the cell culture plate to the incubator (37 &#176;C; 5% CO2 air atmosphere) for an additional 22 h.</li>
		<li>After 22 h, remove the cell culture medium and add 2.0 mL of 18 M&#937; water to the cell monolayer. 
		NOTE: The water will osmotically lyse the cells.</li>
		<li>Harvest the entire cell lysate into standard polypropylene microcentrifuge tubes or similar for subsequent cell protein and cell ferritin analyses.</li>
	</ol>
	</li>
</ol>

<table><tbody><tr><td>0.5 M HCl</td><td>Fisher Scientific</td><td>A508-4 Hydrochloric Acid TraceMetal Grade</td><td></td></tr><tr><td>18 megaohm water</td><td></td><td></td><td>Also known as distilled, deionized water</td></tr><tr><td>3,3&amp;prime;,5-Triiodo-L-thyronine sodium salt</td><td>Sigma Aldrich Co</td><td>T6397</td><td></td></tr><tr><td>6-well plates</td><td>Costar</td><td>3506</td><td>Use for bioassay experiments</td></tr><tr><td>ascorbic acid</td><td>Sigma Aldrich Co</td><td>A0278</td><td></td></tr><tr><td>bile extract</td><td>Sigma Aldrich Co</td><td>B8631</td><td></td></tr><tr><td>Caco-2 cells</td><td>American Type Culture Collection</td><td>HTB-37</td><td>HTB-37 is a common variety.</td></tr><tr><td>Cell culture flasks T225</td><td>Falcon</td><td>353138</td><td></td></tr><tr><td>Cell culture flasks T25</td><td>Corning</td><td>430639</td><td></td></tr><tr><td>Cell culture flasks T75</td><td>Corning</td><td>430641U</td><td></td></tr><tr><td>Chelex-100</td><td>Bio-Rad Laboratories Inc</td><td>142832</td><td>Known as the weak cation exchange resin in the protocol</td></tr><tr><td>collagen</td><td>Corning</td><td>354236</td><td></td></tr><tr><td>dialysis membrane</td><td>Spectrum Laboratories</td><td>Spectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO</td><td>Spectra/Por 7 Pretreated RC Dialysis Tubing 15,000 MWCO</td></tr><tr><td>Dulbecco&amp;rsquo;s Modified Eagle&amp;rsquo;s Medium</td><td>Gibco</td><td>12100046</td><td>DMEM</td></tr><tr><td>epidermal growth factor</td><td>Sigma Aldrich Co</td><td>E4127-5X.1MG</td><td></td></tr><tr><td>fetal bovine serum</td><td>R&amp;amp;D Systems</td><td>S12450</td><td>Optima</td></tr><tr><td>HEPES</td><td>Sigma Aldrich Co</td><td>H3375</td><td></td></tr><tr><td>Hydrocortisone-Water Soluble</td><td>Sigma Aldrich Co</td><td>H0396</td><td></td></tr><tr><td>insert ring</td><td>Corning Costar</td><td>not sold</td><td>Transwell, for 6 well plate, without membrane</td></tr><tr><td>insulin</td><td>Sigma Aldrich Co</td><td>I2643</td><td></td></tr><tr><td>KCl</td><td>Sigma Aldrich Co</td><td>P9333</td><td></td></tr><tr><td>large column</td><td>VWR International</td><td>KT420400-1530</td><td></td></tr><tr><td>Minimum Essential Medium</td><td>Gibco</td><td>41500034</td><td>MEM</td></tr><tr><td>NaCl</td><td>Fisher Scientific</td><td>S271</td><td></td></tr><tr><td>pancreatin</td><td>Sigma Aldrich Co</td><td>P1750</td><td></td></tr><tr><td>PIPES disodium salt</td><td>Sigma Aldrich Co</td><td>Piperazine-1,4-bis(2-ethanesulfonic acid) disodium salt P3768</td><td></td></tr><tr><td>porcine pepsin</td><td>Sigma Aldrich Co</td><td>P6887 or (P7012-25G Sigma</td><td></td></tr><tr><td>silicone o rings</td><td>Web Seal, Inc Rochester NY</td><td>2-215S500</td><td></td></tr><tr><td>sodium bicarbonate</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>Sodium selenite</td><td>Sigma Aldrich Co</td><td>S5261</td><td></td></tr><tr><td>ZellShield</td><td>Minerva Biolabs</td><td>13-0050</td><td>Use at 1% as antibiotic/antimycotic ordered through Thomas Scientific</td></tr></tbody></table>

caco-2 cell bioassay: an in vitro method for measuring iron bioavailability in complex food sources

Source: Glahn, R. P. <a href="https://app.jove.com/v/63859">The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability.</a> J. Vis. Exp. (2022)
This video demonstrates an in vitro technique to assess iron bioavailability in complex food sources. The technique combines the simulation of human digestion along with iron uptake by differentiated Caco-2 cells. Intracellular ferritin formation acts as a marker for cellular iron uptake.

<img alt="Figure 1" src="/files/ftp_upload/21211/21211_Figure1.jpg" /> 
Figure 1: Insert ring setup for Caco-2 cell Fe uptake. (A) Image of Caco-2 cells and insert ring with attached dialysis membrane. (B) Diagram of the overall procedure for in vitro digestion coupled with Caco-2 cell Fe uptake within a single well of the multi-well plate.

Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Source: Glahn, R. P. The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability. J. Vis. Exp. (2022)
This video demonstrates an in vitro ...

Enterocytes are specialized small intestine cells that play a crucial role in nutrient uptake, including iron absorption.
To assess iron bioavailability - the proportion of ingested iron accessible for physiological functions - take an acidified solution containing the food sample. Now, add pepsin - a protein digestion enzyme, from the stomach. At acidic pH, pepsin initiates gastric digestion by breaking down complex dietary proteins into small peptides.
Add sodium bicarbonate to increase the pH and stop gastric digestion. Add a solution containing pancreatic enzymes and bile to simulate intestinal digestion. Transfer the mixture into the upper portion of a two-chamber cell culture system.
The lower chamber contains enterocytes differentiated from Caco-2 - a colon adenocarcinoma cell line. A dialysis membrane separates the upper chamber from the lower one. The membrane allows the diffusion of low molecular weight species.
The pancreatic enzymes-bile mixture in the upper chamber breaks down carbohydrates, proteins, and lipids into smaller subunits - releasing iron in the form of ferric ions.
Bioavailability enhancers in the food convert the released ferric ions to the ferrous state - increasing their solubility. These ions pass through the membrane to reach the lower chamber.
Metal ion transporters in the cell membrane of enterocytes aid in the uptake of ferrous ions. Once inside, iron binds to apoferritin - an iron storage protein resulting in a complex, ferritin. Harvest the cells to quantify ferritin to assess iron bioavailability.

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318 Views. Source: Glahn, R. P. The Caco-2 Cell Bioassay for Measurement of Food Iron Bioavailability. J. Vis. Exp. (2022) This video demonstrates an in vitro technique to assess iron bioavailability in complex food sources. The technique combines the simulation of human digestion along with iron uptake by differentiated Caco-2 cells. Intracellular ferritin formation acts as a marker for cellular iron uptake. 

Video: Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources - Concept

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing a barrier to foreign particles. Impaired epithelial transport (ion, nutrient, or drugs) has been associated with many diseases and can have consequences that extend beyond the normal physiological functions of the transporters, such as by influencing epithelial integrity and the gut microbiome. Understanding the function and regulation of transport proteins is critical for the development of improved therapeutic interventions. The biggest challenge in the study of epithelial transport is developing a suitable model system that recapitulates important features of the native intestinal epithelial cells. Several in vitro cell culture models, such as Caco-2, T-84, and HT-29-Cl.19A cells are typically used in epithelial transport research. These cell lines represent a reductionist approach to modeling the epithelium and have been used in many mechanistic studies, including their examination of epithelial-microbial interactions. However, cell monolayers do not accurately reflect cell-cell interactions and the in vivo microenvironment. Cells grown in 3D have shown to be promising models for drug permeability studies. We show that Caco-2 cells in 3D can be used to study epithelial transporters. It is also important that studies in Caco-2 cells are complemented with other models to rule out cell specific effects and to take into account the complexity of the native intestine. Several methods have been previously used to assess the functionality of transporters, such as everted sac and uptake in isolated epithelial cells or in isolated plasma membrane vesicles. Taking into consideration the challenges in the field with respect to models and the measurement of transport function, we demonstrate here a protocol to grow Caco-2 cells in 3D and describe the use of an Ussing chamber as an effective approach to measure serotonin transport, such as in intact polarized intestinal epithelia.

We describe simple methods to study the regulation of intestinal serotonin transporter (SERT) function and expression using an in vitro cell culture model of Caco-2 cells grown in 3D&#160;and an ex vivo model of mouse intestines. These methods are applicable to the study of other epithelial transporters.

The intestinal epithelium has important transport and barrier functions that play key roles in normal physiological functions of the body while providing ...

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Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a systematic approach combining different methodologies to assess the availability of zinc (Zn) in Atlantic salmon (Salmo salar). Considering that several Zn chemical species can be present in an Atlantic salmon feed, it was hypothesised that Zn availability is influenced by the Zn chemical species present in the feed. Thus, in this study, the first protocol is about how to extract the different Zn chemical species from the feed and to analyze them by a size exclusion chromatography-inductively coupled plasma mass spectroscopy (SEC-ICP-MS) method. Subsequently, an in vitro method was developed to evaluate the solubility of dietary Zn in Atlantic salmon feeds. The third protocol describes the method to study the impact of changing Zn chemical species composition on the uptake of Zn in a fish intestinal epithelial model using a rainbow trout gut cell line (RTgutGC). Together, the findings from the in vitro methods were compared with an in vivo study examining the apparent availability of inorganic and organic sources of Zn supplemented to Atlantic salmon feeds. The results showed that several Zn chemical species can be found in feeds and the efficiency of an organic Zn source depends very much on the amino acid ligand used to chelate Zn. The findings of the in vitro methods had less correlation with that outcome of the in vivo study. Nevertheless, in vitro protocols described in this article provided crucial information regarding Zn availability and its assessment in fish feeds.

This article explains in detail a systematic approach to assess micro-mineral availability in Atlantic salmon. The methodology includes tools and models with increasing biological complexity: (1) chemical speciation analysis, (2) in vitro solubility, (3) uptake studies in cell lines, and (4) in vivo fish studies.&#160;

Assessing the availability of dietary micro-minerals is a major challenge in mineral nutrition of fish species. The present article aims to describe a ...

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been reported that skeletal muscle may increase the extraction of glucose from the blood by up to 50-fold during exercise compared to resting conditions. The increase in muscle glucose uptake during exercise and insulin stimulation is dependent on the translocation of glucose transporter 4 (GLUT4) from intracellular compartments to the muscle cell surface membrane, as well as phosphorylation of glucose to glucose-6-phosphate by hexokinase II. Isolation and incubation of mouse muscles such as m. soleus and m. extensor digitorum longus (EDL) is an appropriate ex vivo model to study the effects of insulin and electrically-induced contraction (a model for exercise) on glucose uptake in mature skeletal muscle. Thus, the ex vivo model permits evaluation of muscle insulin sensitivity and makes it possible to match muscle force production during contraction ensuring uniform recruitment of muscle fibers during measurements of muscle glucose uptake. Moreover, the described model is suitable for pharmacological compound testing that may have an impact on muscle insulin sensitivity or may be of help when trying to delineate the regulatory complexity of skeletal muscle glucose uptake.
Here we describe and provide a detailed protocol on how to measure insulin- and contraction-stimulated glucose uptake in isolated and incubated soleus and EDL muscle preparations from mice using radiolabeled [3H]2-deoxy-D-glucose and [14C]mannitol as an extracellular marker. This allows accurate assessment of glucose uptake in mature skeletal muscle in the absence of confounding factors that may interfere in the intact animal model. In addition, we provide information on metabolic viability of incubated mouse skeletal muscle suggesting that the method&#160;applied possesses some caveats under certain conditions when studying muscle energy metabolism.

Intact regulation of muscle glucose uptake is important for maintaining whole body glucose homeostasis. This protocol presents assessment of insulin- and contraction-stimulated glucose uptake in isolated and incubated mature skeletal muscle when delineating the impact of various physiological interventions on whole body glucose metabolism.

Skeletal muscle is an insulin-responsive tissue and typically takes up most of the glucose that enters the blood after a meal. Moreover, it has been ...

Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Transcript

Caco-2 Cell Bioassay: An In Vitro Method for Measuring Iron Bioavailability in Complex Food Sources

Methods to Study Epithelial Transport Protein Function and Expression in Native Intestine and Caco-2 Cells Grown in 3D

Assessing Mineral Availability in Fish Feeds using Complementary Methods Demonstrated with the Example of Zinc in Atlantic Salmon

Measurement of Insulin- and Contraction-Stimulated Glucose Uptake in Isolated and Incubated Mature Skeletal Muscle from Mice